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Investigating RNA–Protein Interactions in Neisseria meningitidis by RIP-Seq Analysis

  • Nadja Heidrich
  • Saskia Bauriedl
  • Christoph SchoenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1969)

Abstract

Deep sequencing technology has revolutionized transcriptome analyses of both prokaryotes and eukaryotes. RNA-sequencing (RNA-seq), which is based on massively parallel sequencing of cDNAs, has been used to annotate transcript boundaries and has revealed widespread antisense transcription as well as a wealth of novel noncoding transcripts in many bacterial pathogens. Moreover, RNA-seq is nowadays also widely used to comprehensively explore the interaction between RNA-binding proteins and their RNA targets on a genome-wide level in many human-pathogenic bacteria. In particular, immunoprecipitation of an RNA-binding protein (RBP) of interest followed by isolation and analysis of all bound RNAs (RNA immunoprecipitation (RIP)) allows rapid characterization of its RNA regulon. Here, we describe an experimental approach which employs co-immunoprecipitation (coIP) of the RNA-binding chaperone Hfq along with bound RNAs followed by deep-sequencing of co-purified RNAs (RIP-Seq) from a genetically modified strain of Neisseria meningitidis expressing a chromosomally encoded Hfq-3×FLAG protein. This approach allowed us to comprehensively identify both mRNAs and sRNAs as targets of Hfq and served as an excellent starting point for sRNA research in this human pathogenic bacterium.

Key words

RIP-seq N. meningitidis Deep sequencing cDNA library Small RNA identification Hfq-3×FLAG 

Notes

Acknowledgments

The work in the Vogel lab was funded by DFG Grant Vo875/7-2.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Nadja Heidrich
    • 1
  • Saskia Bauriedl
    • 2
  • Christoph Schoen
    • 3
    Email author
  1. 1.Institute for Molecular Infection Biology (IMIB)University of WürzburgWürzburgGermany
  2. 2.Institute for Molecular Infection Biology (IMIB) and Institute for Hygiene and Microbiology (IHM)University of WürzburgWürzburgGermany
  3. 3.Institute for Hygiene and Microbiology (IHM)University of WürzburgWürzburgGermany

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